Frank E. Stockdale

DNA synthesis and myogenesis

Abstract Differentiating muscle cells synthesizing myosin, the meromyosins, and actin do not concurrently synthesize DNA. Presumptive myoblasts which synthesize DNA do not concurrently synthesize myosin, the meromyosins or actin. The multinucleated skeletal muscle fiber is the product of cell fusion.

The importance of the lumpectomy surgical margin status in long term results of breast conservation

Background. The impact of the surgical margin status on long term local control rates for breast cancer in women treated with lumpectomy and radiation therapy is unclear.

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.

Myogenic cell lineages

For many years the mechanisms by which skeletal muscles in higher vertebrates come to be composed of diverse fiber types distributed in distinctive patterns has interested cell and developmental biologists. The fiber composition of skeletal muscles varies from class to class and from muscle to muscle within the vertebrates. The developmental basis for these events is the subject of this review. Because an individual multinucleate vertebrate skeletal muscle fiber is formed by the fusion of many individual myoblasts, more attention, in recent times, has been directed toward the origins and differences among myoblasts, and more emphasis has been placed on the lineal relationship of myoblasts to fibers. This is a review of studies related to the concepts of myogenic cell lineage in higher vertebrate development with emphases on some of the most challenging problems of myogenesis including the embryonic origins of myogenic precursor cells, the mechanisms of fiber type diversity and patterning, the distinctions among myoblasts during myogenesis, and the current hypotheses of how a variety of factors, intrinsic and extrinsic to the myoblast, determine the definitive phenotype of a muscle fiber.

New models and online calculator for predicting non-sentinel lymph node status in sentinel lymph node positive breast cancer patients

BackgroundCurrent practice is to perform a completion axillary lymph node dissection (ALND) for breast cancer patients with tumor-involved sentinel lymph nodes (SLNs), although fewer than half will have non-sentinel node (NSLN) metastasis. Our goal was to develop new models to quantify the risk of NSLN metastasis in SLN-positive patients and to compare predictive capabilities to another widely used model.MethodsWe constructed three models to predict NSLN status: recursive partitioning with receiver operating characteristic curves (RP-ROC), boosted Classification and Regression Trees (CART), and multivariate logistic regression (MLR) informed by CART. Data were compiled from a multicenter Northern California and Oregon database of 784 patients who prospectively underwent SLN biopsy and completion ALND. We compared the predictive abilities of our best model and the Memorial Sloan-Kettering Breast Cancer Nomogram (Nomogram) in our dataset and an independent dataset from Northwestern University.Results285 patients had positive SLNs, of which 213 had known angiolymphatic invasion status and 171 had complete pathologic data including hormone receptor status. 264 (93%) patients had limited SLN disease (micrometastasis, 70%, or isolated tumor cells, 23%). 101 (35%) of all SLN-positive patients had tumor-involved NSLNs. Three variables (tumor size, angiolymphatic invasion, and SLN metastasis size) predicted risk in all our models. RP-ROC and boosted CART stratified patients into four risk levels. MLR informed by CART was most accurate. Using two composite predictors calculated from three variables, MLR informed by CART was more accurate than the Nomogram computed using eight predictors. In our dataset, area under ROC curve (AUC) was 0.83/0.85 for MLR (n = 213/n = 171) and 0.77 for Nomogram (n = 171). When applied to an independent dataset (n = 77), AUC was 0.74 for our model and 0.62 for Nomogram. The composite predictors in our model were the product of angiolymphatic invasion and size of SLN metastasis, and the product of tumor size and square of SLN metastasis size.ConclusionWe present a new model developed from a community-based SLN database that uses only three rather than eight variables to achieve higher accuracy than the Nomogram for predicting NSLN status in two different datasets.

5-Bromodeoxyuridine: Effect on Myogenesis in vitro

Presumptive myoblasts, obtained by treating muscle from 11-day chick embryos with trypsin, multiply in vitro. On the 4th or 5th day in culture they abruptly fuse, form long multinucleated myotubes, and begin to synthesize myosin. Cultured cells exposed to 5-bromodeoxyuridine incorporate this analog of thymidine into their DNA. Cells with such falsified DNA are reversibly inhibited from forming myotubes and synthesizing myosin; such cells, however, continue to synthesize the various species of molecules required for cell multiplication.

Reduced cardiotoxicity of doxorubicin delivered on a weekly schedule. Assessment by endomyocardial biopsy.

Endomyocardial biopsy was done 119 times in 98 patients receiving doxorubicin therapy once every 3 weeks and 41 times in 27 patients receiving doxorubicin therapy weekly. Factors contributing to the degree of anthracycline-induced endomyocardial injury were evaluated. Neither age, sex, type of malignancy, concomitant use of other chemotherapeutic agents including cyclophosphamide, nor history of cardiac disease or hypertension influenced the extent of the endomyocardial injury. The dose of doxorubicin (p = 0.0001) and the schedule (weekly versus 3 weekly) (p = 0.0020) independently predicted the degree of endomyocardial damage in multivariate analyses. Previous cardiac irradiation had borderline significance (p = 0.074) in predicting endomyocardial damage in this analysis. Doxorubicin therapy administered on a weekly schedule is associated with less anthracycline-induced cardiac damage than is doxorubicin therapy delivered in the conventional, 3-weekly schedule.

Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2(+) breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the FcγRIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.

Behavior of cancer patients: a randomized study of the effects of education and peer support groups.

TWO PROSPECTIVE, CONTROLLED STUDIES were conducted to determine if psychological and social functioning could be enhanced in patients with Hodgkins disease by either education or participation in a peer support therapy group. Eighty-one patients were evaluated with the Cancer Patient Behavior Scale prior to and following intervention. Following education, patients experienced significant improvement in the frequency of Anxiety, Treatment Problems, Depression, and Life Disruption (p ≤0.1) compared to a control group. Following participation in peer support groups, patients showed no improvement in any of 10 areas of life functioning. Thus education represents an effective, efficient, and inexpensive means of improving psychological and social behavior in patients with Hodgkins disease. Participation in this peer support therapy group did not result in significant behavior change.

Skeletal muscle satellite cell diversity: Satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.